CN106148243A - Invertase arthrobacterium and application thereof are produced in one strain - Google Patents
Invertase arthrobacterium and application thereof are produced in one strain Download PDFInfo
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- CN106148243A CN106148243A CN201610753885.1A CN201610753885A CN106148243A CN 106148243 A CN106148243 A CN 106148243A CN 201610753885 A CN201610753885 A CN 201610753885A CN 106148243 A CN106148243 A CN 106148243A
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- 108010051210 beta-Fructofuranosidase Proteins 0.000 title claims abstract description 25
- 235000011073 invertase Nutrition 0.000 title claims abstract description 23
- 239000001573 invertase Substances 0.000 title abstract 3
- 108090000790 Enzymes Proteins 0.000 claims abstract description 24
- 102000004190 Enzymes Human genes 0.000 claims abstract description 24
- FVVCFHXLWDDRHG-UPLOTWCNSA-N (2s,3r,4s,5r,6r)-2-[(2r,3s,4r,5r,6r)-6-[(2s,3s,4s,5r)-3,4-dihydroxy-2,5-bis(hydroxymethyl)oxolan-2-yl]oxy-4,5-dihydroxy-2-(hydroxymethyl)oxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)[C@@H](CO)O1 FVVCFHXLWDDRHG-UPLOTWCNSA-N 0.000 claims abstract description 21
- 241000186073 Arthrobacter sp. Species 0.000 claims abstract description 20
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 15
- 229930006000 Sucrose Natural products 0.000 claims abstract description 15
- 239000005720 sucrose Substances 0.000 claims abstract description 15
- 244000005700 microbiome Species 0.000 claims abstract 3
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 28
- 239000007788 liquid Substances 0.000 claims description 26
- 239000007787 solid Substances 0.000 claims description 22
- 238000000855 fermentation Methods 0.000 claims description 20
- 230000004151 fermentation Effects 0.000 claims description 20
- 239000001963 growth medium Substances 0.000 claims description 18
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 16
- 229940041514 candida albicans extract Drugs 0.000 claims description 14
- 229910000388 diammonium phosphate Inorganic materials 0.000 claims description 14
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 14
- 239000012138 yeast extract Substances 0.000 claims description 14
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 claims description 13
- 235000019838 diammonium phosphate Nutrition 0.000 claims description 13
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 13
- 238000000926 separation method Methods 0.000 claims description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 239000002609 medium Substances 0.000 claims description 12
- 239000012530 fluid Substances 0.000 claims description 11
- 239000000243 solution Substances 0.000 claims description 11
- 238000011218 seed culture Methods 0.000 claims description 10
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 8
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 8
- 239000008101 lactose Substances 0.000 claims description 8
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 8
- 239000008103 glucose Substances 0.000 claims description 7
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 7
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 7
- 239000001888 Peptone Substances 0.000 claims description 6
- 108010080698 Peptones Proteins 0.000 claims description 6
- 238000004140 cleaning Methods 0.000 claims description 6
- 239000000306 component Substances 0.000 claims description 6
- 239000012533 medium component Substances 0.000 claims description 6
- 235000019319 peptone Nutrition 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 5
- 230000003068 static effect Effects 0.000 claims description 5
- 229910019142 PO4 Inorganic materials 0.000 claims description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 3
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 3
- 239000010452 phosphate Substances 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 239000008363 phosphate buffer Substances 0.000 claims description 2
- 238000005057 refrigeration Methods 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 8
- 231100000350 mutagenesis Toxicity 0.000 abstract description 6
- 238000002703 mutagenesis Methods 0.000 abstract description 6
- 239000002689 soil Substances 0.000 abstract description 6
- 238000005516 engineering process Methods 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 2
- 231100000219 mutagenic Toxicity 0.000 abstract description 2
- 230000003505 mutagenic effect Effects 0.000 abstract description 2
- 230000001580 bacterial effect Effects 0.000 description 9
- 230000002255 enzymatic effect Effects 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 230000002906 microbiologic effect Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000005352 clarification Methods 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 239000013028 medium composition Substances 0.000 description 2
- 230000035790 physiological processes and functions Effects 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 235000019605 sweet taste sensations Nutrition 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- CHUGKEQJSLOLHL-UHFFFAOYSA-N 2,2-Bis(bromomethyl)propane-1,3-diol Chemical compound OCC(CO)(CBr)CBr CHUGKEQJSLOLHL-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000186000 Bifidobacterium Species 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N Furan Chemical compound C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 206010054949 Metaplasia Diseases 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 235000008429 bread Nutrition 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 235000021185 dessert Nutrition 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 235000013376 functional food Nutrition 0.000 description 1
- 235000021255 galacto-oligosaccharides Nutrition 0.000 description 1
- 150000003271 galactooligosaccharides Chemical class 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000008944 intestinal immunity Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 230000015689 metaplastic ossification Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000000844 transformation Methods 0.000 description 1
- -1 β-fructofuranose Glycosides Chemical class 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/06—Arthrobacter
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2431—Beta-fructofuranosidase (3.2.1.26), i.e. invertase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01026—Beta-fructofuranosidase (3.2.1.26), i.e. invertase
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Abstract
The present invention relates to a strain and produce invertase arthrobacterium and application thereof.One strain arthrobacterium (Arthrobacter sp.) BLCY 004, on August 16th, 2016 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, preserving number CGMCC No.12855.The present invention isolates arthrobacterium from soil, through the mutagenic treatment technology such as ultraviolet mutagenesis, NTG mutant treatment, finally obtain the named BLCY of superior strain 004 of high yield invertase, its enzyme is lived and is reached 1100U/ml, improve more than 50% relative to conventional beta fructofuranoside enzyme activity, it is applied to be greatly improved, during lactosucrose produces, the ability that sucrose inversion becomes lactosucrose, significantly reduce production cost.
Description
Technical field
The present invention relates to a strain and produce saccharase arthrobacterium product and application thereof, belong to technical field of biotechnology.
Background technology
Lactosucrose is a kind of functional oligose, has promotion intestinal bifidobacteria and breeds, regulating intestinal canal Tiny ecosystem,
Improving the special physiological functions such as intestinal immunity, sugariness is the 30% of sucrose, and sweet taste characteristic is similar to sucrose, and sweet taste quality is each
In kind of compound sugar optimal, therefore can apply in food industry without worrying that it produces impact to product special flavour.Business
The lactosucrose that metaplasia is produced, due to containing other compositions such as sucrose, lactose, thus sugariness is slightly higher.Oligomeric with other
Sugar is compared, and lactosucrose has higher stability to acid, heat, and its stability is similar to sucrose, stable in neutral conditions,
Relatively more stable in acid condition, the lower 80 DEG C of heating 2h of lower 80 DEG C of heating 2h and pH 3.0 of pH 7.0, occur all hardly to divide
Solving, under conditions of pH 4.5, its heating-up temperature is even up to 120 DEG C.Lactosucrose also has high moisture retention, can make
Food keeps moistening, and the starch food products such as bread, dessert can be prevented aging, extends Food Shelf-life.
Investigation in 2005 shows, lactosucrose has become the Japanese the third-largest functional oligose consumer goods.Permitted in recent years
Many researchers find that lactosucrose has many specific physiological functions, are expected to become continue lactosucrose, galactooligosaccharide
Another functional food additives approved by the whole world rear.
Enzymatic clarification is the main path of industrialized production lactosucrose, and the productivity of synthesizing lactosucrose by enzyme method is low by one
Being directly the important problem of restriction enzymatic clarification development, therefore, the saccharase finding a kind of enzymatic activity high becomes solution
The key of lactosucrose production bottleneck.
Chinese patent literature CN101624582A (application number 200910115791.1) discloses a kind of arthrobacterium and produces β-furan
Muttering the fermentation medium of fructosidase and fermentation process, belonging to biotechnology engineering field, fermentation medium composition is: sucrose
3～7g/L, beef extract 25～40g/L, yeast extract 2～3g/L, (NH4)2HPO48～12g/L, KH2PO40.5～1g/L,
MgSO4·7H2O0.1～0.5g/L, Yu Weishui；It first by actication of culture, cultivation, is subsequently adding above-mentioned fermentation medium, initial pH
Value is 6.0～8.0, and fermentation temperature is 25～45 DEG C, and inoculum concentration is 1～4%；The present invention is significantly better than the culture medium of prior art
Formula, bacterial strain biomass is high, yield of enzyme is high, enzymatic activity is high.
Technique scheme is started with from culture environment, and the culture medium obtaining and fermentation process can improve production of enzyme, but make
The principal element that about production of enzyme and enzyme are lived is in terms of bacterial strain, therefore finds production of enzyme height, enzyme high bacterial strain alive, becomes current
Study hotspot.
Content of the invention
The present invention is directed to the deficiencies in the prior art, provide a strain to produce saccharase arthrobacterium and application thereof.
Another object of the present invention is to provide application in producing lactosucrose for this pole bacterial strain, by this bacterial strain institute
Produce saccharase and there is the ability of high synthesizing lactosucrose, production cost can be significantly reduced.
Technical scheme is as follows:
One strain arthrobacterium (Arthrobacter sp.) BLCY-004, is preserved in China Microbiological bacterium on August 16th, 2016
Plant preservation administration committee common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences is micro-
Biological study institute, preserving number CGMCC No.12855.
The original strain of arthrobacterium of the present invention (Arthrobacter sp.) BLCY-004 is located away from Dezhou, Shandong hundred
Soil near the workshop of Long Chuan garden, and obtain after mutagenesis.This bacterial strain is creamy white, translucent, size about 2～4 μ
M × 1.8～3.5 μm.Gram's staining is positive, rod-short, single or arrange in pairs, along with shaft-like, ball in cultivation cycle
The change of shape form.This bacterial strain can high yield saccharase, generate lactosucrose conversion ratio can reach more than 30%,
It is greatly improved in applying transformations to produce and generates the ability of lactosucrose with sucrose and lactose for substrate, reduce and produce into
This, contribute to the popularization of lactosucrose product.
The cultural method of above-mentioned arthrobacterium (Arthrobacter sp.) BLCY-004, step is as follows:
(1) take arthrobacterium (Arthrobacter sp.) BLCY-004 to be inoculated in solid medium, at the bar of 28-35 DEG C
Under part, cultivate 24-48h, prepare activated strains；
(2) take the activated strains that step (1) prepares, be inoculated in seed culture medium, under conditions of 28～35 DEG C, propagation
Cultivate 24-48h, obtain seed liquor；
(3) taking the seed liquor that step (2) prepares, the ratio of 1～10% is inoculated in fermentation medium by volume, 28
～35 DEG C expand cultivation 36～60h, obtain thalline zymotic fluid.
According to currently preferred, the seed culture medium component in described step (2) is as follows, is all weight percentage:
Glucose 2%, yeast extract 1.5%, potassium dihydrogen phosphate 0.2%, diammonium hydrogen phosphate 0.6%, epsom salt
0.01%, pH6.0～7.2.
According to currently preferred, the fermentation medium component in described step (3) is as follows, is all weight percentage:
Sucrose 4%, lactose 1%, yeast extract 1.2%, peptone 0.8%, epsom salt 0.1%, diammonium hydrogen phosphate
0.4%, pH 6.0～7.2.
Solid medium in described step (1) is this area Conventional solid culture medium, such as LB culture medium etc..
Above-mentioned arthrobacterium (Arthrobacter sp.) application in preparing saccharase for the BLCY-004.
Above-mentioned application, step is as follows:
A () takes the thalline fermentation liquor separation of solid and liquid of above-mentioned preparation, wash thalline, merges and separates liquid and cleaning solution, prepares
Crude enzyme liquid；
B (), by adding solid sodium chloride in crude enzyme liquid prepared for step (a), prepares the mixed solution of 0.1～0.5mol/L,
Refrigerate static 2h, separation of solid and liquid, take supernatant, add solid sodium chloride, make solution reach the ammonium sulfate saturation degree of 90%, cold
Hide static 2h, separation of solid and liquid, take precipitation, be dissolved in the phosphate-buffered of pH the 7.8th, 0.05mol/L, prepare β-fructofuranose
Glycosides enzyme.
According to currently preferred, that the washing thalline of described step (a) is employing pH the 7.0th, concentration 40mmol/L phosphoric acid
Buffer solution is resuspended, then through separation of solid and liquid, takes liquid, prepares cleaning solution.
According to currently preferred, described step (a), the separation of solid and liquid in (b) are 4 DEG C, 4000r/min condition
Under, centrifugal 40min.
Application in preparing lactosucrose for the saccharase of above-mentioned preparation.
Beneficial effect
The present invention isolates arthrobacterium from soil, through the mutagenic treatment such as ultraviolet mutagenesis, NTG mutant treatment
Technology, finally obtains the named BLCY-004 of superior strain of high yield saccharase, and its enzyme is lived and reached 1100U/ml, phase
More than 50% is improved for tradition saccharase vigor, is applied to be greatly improved sucrose during lactosucrose produces and turns
The ability of chemical conversion lactosucrose, significantly reduces production cost.
Detailed description of the invention
Below in conjunction with embodiment, technical scheme is further elaborated, but institute of the present invention protection domain is not limited to
This.
Embodiment 1
One strain arthrobacterium (Arthrobacter sp.) BLCY-004, is preserved in China Microbiological bacterium on August 16th, 2016
Plant preservation administration committee common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences is micro-
Biological study institute, preserving number CGMCC No.12855.
The screening process of above-mentioned arthrobacterium (Arthrobacter sp.) BLCY-004 is as follows:
(1) enrichment culture
Choose the soil near the workshop of Long Chuan garden, Dezhou, Shandong hundred, remove table soil with little scoop, take overhead 5-
Soil at 15cm about 10g, dilutes 10 times with sterilized water, adds culture medium to carry out enrichment culture, medium component: glucose
2%, yeast extract 1.5%, potassium dihydrogen phosphate 0.2%, diammonium hydrogen phosphate 0.6%, epsom salt 0.01%, pH is 6.5；Cultivate
Temperature is 28-35 DEG C, cultivates 36h.
(2) Pure strain separation
Use line partition method, take a Boiling tube filling 5ml sterilized water, take the bacterium after enrichment culture in step (1)
Liquid 2ml puts into and wherein dilutes, abundant vibrating dispersion, with oese with sterile working picking dilution one ring first at plating medium
While doing parallel scribing 3-4 bar for the first time, being rotated further by about 60 degree of angles of culture dish, residue on oese being burnt, after cooling
Do second time line with a scribble method, take turns doing third time and the 4th line with method.Line finishes, and covers ware lid, will
Culture dish is inverted, and cultivates after 36h for 30 DEG C, the single colony inoculation of picking on 10 slant mediums, numbering 01-10 respectively.
Be inoculated in 01-10 inclined-plane seed in Shake flask medium cultivation 32 DEG C cultivate 36h, to 01-10 shake flask fermentation liquid β-
Fructofuranosidase enzyme is lived and is measured, and No. 04 shaking flask enzyme is lived the highest, reaches 505U/ml.
Plating medium composition: glucose 2%, yeast extract 1.5%, potassium dihydrogen phosphate 0.2%, diammonium hydrogen phosphate 0.6%,
Epsom salt 0.01%, agar 2%, pH is pH6.0-7.2.
Shaking culture based component: sucrose 4%, lactose 1%, yeast extract 1.2%, peptone 0.8%, epsom salt
0.1%, diammonium hydrogen phosphate 0.4%, pH 6.0-7.2.
(3) mutagenesis screening
Carry out ultraviolet mutagenesis to No. 04 bacterial classification, ultraviolet mutagenesis uses 15W ultraviolet lamp 20cm to irradiate, and irradiation time is
150s, finally gives the named BLCY-004 of superior strain of high yield saccharase, and its enzyme is lived and reached 1100U/ml.
Enzyme is lived and is defined as follows: at pH 6.0,40 DEG C using sucrose as substrate react when, per minute make reducing power increase
The enzyme amount of the amount adding the D-Glucose being equivalent to 2 μm of ol schedules be 1 unit of activity (U).
Embodiment 2
The cultural method of arthrobacterium (Arthrobacter sp.) BLCY-004 described in embodiment 1, step is as follows:
(1) take arthrobacterium (Arthrobacter sp.) BLCY-004 to be inoculated in LB culture medium, under conditions of 30 DEG C,
Activation culture 30h, prepares activated strains；
(2) take the activated strains that step (1) prepares, be inoculated in seed culture medium, under conditions of 32 DEG C, Multiplying culture
36h, prepares seed liquor；
Described seed culture medium component is as follows, is all weight percentage:
Glucose 2%, yeast extract 1.5%, potassium dihydrogen phosphate 0.2%, diammonium hydrogen phosphate 0.6%, epsom salt
0.01%, pH6.5.
(3) taking the seed liquor that step (2) prepares, the ratio of 1% is inoculated in fermentation medium by volume, at 30 DEG C,
Expand and cultivate 35h, obtain thalline zymotic fluid；
Described fermentation medium component is as follows, is all weight percentage:
Sucrose 4%, lactose 1%, yeast extract 1.2%, peptone 0.8%, epsom salt 0.1%, diammonium hydrogen phosphate
0.4%, pH 6.5.
After testing, the cell concentration of above-mentioned prepared thalline zymotic fluid is 6.5 × 1010cfu/ml。
Embodiment 3
The cultural method of arthrobacterium (Arthrobacter sp.) BLCY-004 described in embodiment 1, step is as follows:
(1) take arthrobacterium (Arthrobacter sp.) BLCY-004 to be inoculated in LB culture medium, under conditions of 31 DEG C,
Activation culture 36h, prepares activated strains；
(2) take the activated strains that step (1) prepares, be inoculated in seed culture medium, under conditions of 32 DEG C, Multiplying culture
36h, prepares seed liquor；
Described seed culture medium component is as follows, is all weight percentage:
Glucose 2%, yeast extract 1.5%, potassium dihydrogen phosphate 0.2%, diammonium hydrogen phosphate 0.6%, epsom salt
0.01%, pH 7.0.
(3) taking the seed liquor that step (2) prepares, the ratio of 5% is inoculated in fermentation medium by volume, at 30 DEG C,
Expand and cultivate 40h, obtain thalline zymotic fluid；
Described fermentation medium component is as follows, is all weight percentage:
Sucrose 4%, lactose 1%, yeast extract 1.2%, peptone 0.8%, epsom salt 0.1%, diammonium hydrogen phosphate
0.4%, pH 6.8.
After testing, the cell concentration of above-mentioned prepared thalline zymotic fluid is 6.0 × 1010cfu/ml。
Embodiment 4
The cultural method of arthrobacterium (Arthrobacter sp.) BLCY-004 described in embodiment 1, step is as follows:
(1) take arthrobacterium (Arthrobacter sp.) BLCY-004 to be inoculated in LB culture medium, under conditions of 35 DEG C,
Activation culture 48h, prepares activated strains；
(2) take the activated strains that step (1) prepares, be inoculated in seed culture medium, under conditions of 35 DEG C, Multiplying culture
48h, prepares seed liquor；
Described seed culture medium component is as follows, is all weight percentage:
Glucose 2%, yeast extract 1.5%, potassium dihydrogen phosphate 0.2%, diammonium hydrogen phosphate 0.6%, epsom salt
0.01%, pH 7.2.
(3) taking the seed liquor that step (2) prepares, the ratio of 10% is inoculated in fermentation medium by volume, at 35 DEG C,
Expand and cultivate 48h, obtain thalline zymotic fluid；
Described fermentation medium component is as follows, is all weight percentage:
Sucrose 4%, lactose 1%, yeast extract 1.2%, peptone 0.8%, epsom salt 0.1%, diammonium hydrogen phosphate
0.4%, pH 7.2.
After testing, the cell concentration of above-mentioned prepared thalline zymotic fluid is 7.0 × 1010cfu/ml。
Embodiment 5
Arthrobacterium (Arthrobacter sp.) application in preparing saccharase for the BLCY-004.
Above-mentioned application, step is as follows:
A centrifugal 40min under the conditions of the thalline fermentation liquor 4 DEG C of () Example 2～4 preparation respectively, 4000r/min, receives
Collection separates liquid, is precipitated as thalline, and the phosphate buffer by pH the 7.0th, concentration 40mmol/L is resuspended, through 4 DEG C, 10000r/min bar
Centrifuge 40min under part, collect cleaning solution, merge and separate liquid and cleaning solution, prepare crude enzyme liquid；
B crude enzyme liquid prepared for step (a) is added solid sodium chloride by (), prepare the mixed solution of 0.1-0.5mol/L, cold
Hide static 2h, separation of solid and liquid, take supernatant, add solid sodium chloride, make solution reach the ammonium sulfate saturation degree of 90%, refrigeration
Static 2h, separation of solid and liquid, take precipitation, be dissolved in the phosphate-buffered of pH the 7.8th, 0.05mol/L, prepare β-fructofuranoside
Enzyme.
After testing, in embodiment 2, every milliliter of zymotic fluid can produce 6000 μ g saccharases, every in embodiment 3
Milliliter zymotic fluid can produce 5800ug saccharase, in embodiment 4 every milliliter of zymotic fluid can produce 6300 μ g β-
Fructofuranosidase.
Above-mentioned saccharase is configured to β-transfructosylase solution that concentration is 15000 μ g/ml, through inspection
Surveying, enzyme is lived and is reached 1100U/ml；
The produced saccharase of No. 04 arthrobacterium original strain of detection under the conditions of same concentrations, it is 505U/ that enzyme is lived
ml。
The above results be can be seen that herein described arthrobacterium (Arthrobacter sp.) BLCY-004 bacterial strain is produced
Saccharase enzyme is lived and is far above original starting strain.
Claims (9)
1. a strain arthrobacterium (Arthrobacter sp.) BLCY-004, is preserved in Chinese microorganism strain on August 16th, 2016
Preservation administration committee common micro-organisms center, address: the micro-life of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences
Thing research institute, preserving number CGMCC No.12855.
2. the cultural method of arthrobacterium described in claim 1 (Arthrobacter sp.) BLCY-004, it is characterised in that step
As follows:
(1) take arthrobacterium (Arthrobacter sp.) BLCY-004 to be inoculated in solid medium, the condition of 28-35 DEG C
Under, cultivate 24-48h, prepare activated strains；
(2) take the activated strains that step (1) prepares, be inoculated in seed culture medium, under conditions of 28～35 DEG C, Multiplying culture
24-48h, obtains seed liquor；
(3) taking the seed liquor that step (2) prepares, the ratio of 1～10% is inoculated in fermentation medium by volume, 28～35
DEG C expand cultivation 36～60h, obtain thalline zymotic fluid.
3. cultural method as claimed in claim 2, it is characterised in that the seed culture medium component in described step (2) is as follows,
It all is weight percentage:
Glucose 2%, yeast extract 1.5%, potassium dihydrogen phosphate 0.2%, diammonium hydrogen phosphate 0.6%, epsom salt 0.01%,
PH6.0～7.2.
4. cultural method as claimed in claim 2, it is characterised in that the fermentation medium component in described step (3) is as follows,
It all is weight percentage:
Sucrose 4%, lactose 1%, yeast extract 1.2%, peptone 0.8%, epsom salt 0.1%, diammonium hydrogen phosphate 0.4%,
PH 6.0～7.2.
5. arthrobacterium described in claim 1 (Arthrobacter sp.) BLCY-004 answering in preparing saccharase
With.
6. apply as claimed in claim 5, it is characterised in that step is as follows:
A () takes the thalline fermentation liquor separation of solid and liquid of above-mentioned preparation, wash thalline, merges and separates liquid and cleaning solution, prepares thick enzyme
Liquid；
B () will add solid sodium chloride in crude enzyme liquid prepared for step (a), prepare the mixed solution of 0.1～0.5mol/L, refrigeration
Static 2h, separation of solid and liquid, take supernatant, add solid sodium chloride, make solution reach the ammonium sulfate saturation degree of 90%, refrigerate quiet
Only 2h, separation of solid and liquid, take precipitation, be dissolved in the phosphate-buffered of pH the 7.8th, 0.05mol/L, prepares saccharase.
7. apply as claimed in claim 6, it is characterised in that the washing thalline of described step (a) is for using pH the 7.0th, concentration
The phosphate buffer of 40mmol/L is resuspended, then through separation of solid and liquid, takes liquid, prepares cleaning solution.
8. apply as claimed in claim 6, it is characterised in that described step (a), the separation of solid and liquid in (b) be 4 DEG C,
Under the conditions of 4000r/min, centrifugal 40min.
9. application in preparing lactosucrose for the saccharase of claim 5 preparation.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108660129A (en) * | 2018-04-28 | 2018-10-16 | 江南大学 | The method that sol-gal process fixes saccharase and the double enzymes of glucose oxidase |
| CN110564629A (en) * | 2019-07-04 | 2019-12-13 | 山东百龙创园生物科技股份有限公司 | trichoderma reesei and culture method and application thereof |
| WO2021000963A1 (en) * | 2019-07-04 | 2021-01-07 | 山东百龙创园生物科技股份有限公司 | Trichoderma reesei strain, culture method therefor, and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101624582A (en) * | 2009-07-30 | 2010-01-13 | 南昌大学 | Fermentation medium for Beta-fructofuranosidase from arthrobacter and fermentation method thereof |
| CN101775423A (en) * | 2010-02-08 | 2010-07-14 | 南昌大学 | Method for synthesizing lactosucrose by enzyme method |
| CN102168028A (en) * | 2010-02-26 | 2011-08-31 | 江南大学 | Arthrobacter mutant strain, method for producing lactase from mutant strain and method for preparing lactulose by using lactase |
| CN103045561A (en) * | 2012-12-17 | 2013-04-17 | 宁夏乙征生物工程有限公司 | Solid state fermentation production method for beta-D-fructofuranosidase |
| CN104073456A (en) * | 2014-07-10 | 2014-10-01 | 江南大学 | Bacterial strain for producing levansucrase and method for producing lactosucrose by utilizing lavansucrase |
-
2016
- 2016-08-29 CN CN201610753885.1A patent/CN106148243B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101624582A (en) * | 2009-07-30 | 2010-01-13 | 南昌大学 | Fermentation medium for Beta-fructofuranosidase from arthrobacter and fermentation method thereof |
| CN101775423A (en) * | 2010-02-08 | 2010-07-14 | 南昌大学 | Method for synthesizing lactosucrose by enzyme method |
| CN102168028A (en) * | 2010-02-26 | 2011-08-31 | 江南大学 | Arthrobacter mutant strain, method for producing lactase from mutant strain and method for preparing lactulose by using lactase |
| CN103045561A (en) * | 2012-12-17 | 2013-04-17 | 宁夏乙征生物工程有限公司 | Solid state fermentation production method for beta-D-fructofuranosidase |
| CN104073456A (en) * | 2014-07-10 | 2014-10-01 | 江南大学 | Bacterial strain for producing levansucrase and method for producing lactosucrose by utilizing lavansucrase |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108660129A (en) * | 2018-04-28 | 2018-10-16 | 江南大学 | The method that sol-gal process fixes saccharase and the double enzymes of glucose oxidase |
| CN108660129B (en) * | 2018-04-28 | 2021-11-30 | 江南大学 | Method for immobilizing beta-fructofuranosidase and glucose oxidase double enzymes by sol-gel method |
| CN110564629A (en) * | 2019-07-04 | 2019-12-13 | 山东百龙创园生物科技股份有限公司 | trichoderma reesei and culture method and application thereof |
| WO2021000963A1 (en) * | 2019-07-04 | 2021-01-07 | 山东百龙创园生物科技股份有限公司 | Trichoderma reesei strain, culture method therefor, and application thereof |
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